ARTICLE TOOLS

Abstract

Semliki Forest virus (SFV) vectors have been developed to provide a convenient system to express protein-encoding sequences in virtually any animal cell. This unit presents two strategies for protein expression using SFV vectors. In both cases the protein-coding sequence of interest is cloned into a plasmid vector, which is subsequently used to produce recombinant RNA in vitro. This RNA, which is of positive polarity, is transfected into cells and there is amplified by virtue of its self-encoded RNA replicase. The same replicase also produces a shorter RNA species that encodes the protein of interest. In the first protocol, cells are transfected (either by electroporation or liposome-mediated transfection) and directly analyzed for expression of the heterologous protein. Accompanying support protocols provide methods for checking expression and transfection through galactosidase assays of transfected cells and cell lysates. The other strategy employs in vivo packaging of the RNA into SFV particles; recombinant RNA is cotransfected with a special helper RNA that codes for the structural proteins needed for virus assembly. SFV particles carrying only recombinant RNA are formed and are used to infect cells for analysis of protein expression. Accompanying support protocols describe methods for titrating and purifying recombinant virus stocks. Although the protocols presented here are designed for use with BHK (baby hamster kidney) cells, the virus has a very broad host range and can be used with many different cell types.